Klebsiella pneumoniae is an opportunistic pathogen frequently implicated in respiratory and urinary tract infections of hospitalized patients. In addition, it is a leading cause of pneumonia in chronic alcoholics and has also been associated with community acquired pneumonia. Infections are particularly difficult to treat since most clinical isolates exhibit resistance to several antibiotics leading to treatment failure. In the human respiratory tract it has been suggested that K. pneumoniae can exist as a biofilm growing on lung tissue. These bacteria produce copious amounts of an acidic polysaccharide capsule that has been shown to possess antiphagocytic properties and the capsule is believed to be an important virulence factor. However, relatively little is known about other virulence determinants produced by these bacteria which allow them to colonize and grow on perturbed epithelial tissues, invade into the bloodstream and successfully overcome host defense mechanisms. In this proposal we describe studies to continue our investigations into the molecular pathogenetic mechanisms of K. pneumoniae. We will use flow-through biofilm chambers coated with human-derived extracellular matrices to determine the role of fimbrial types and other gene products in forming a biofilm on these biotic surfaces. In complementary studies a murine model of acute infection will be utilized to examine the role of putative virulence factors in vivo. In many other pathogenic bacteria the genetic regulation of virulence factor expression involves a complex regulatory network or regulon. We describe in this proposal two series of studies to examine regulators that play an important role in influencing the ability of the bacteria to cause infections in vivo and grow on human matrices in vitro. The targets of these regulatory genes will be determined. In addition, like many other enteric bacteria, K. pneumoniae posses the ability to produce several different types of fimbriae or pili. The coordinate production of distinct fimbrial types has not been investigated in detail in any bacterial species. The isolation of mutations that cause alteration in at least two types of fimbrial expression will allow us to begin to characterize the fimbrial regulon. In summary the aims of this proposal are to elucidate the role of fimbriae in K. pneumoniae pathogenesis, to investigate the regulatory pathways affecting virulence, and to define virulence gene expression during biofilm growth. PUBLIC HEALTH RELEVANCE: This project investigates mechanisms used by the bacterium Klebsiella pneumoniae to cause infections of the airways. These bacteria frequently cause airway infections in hospitalized patients,